Takumi Ohishi

Developmental Exposure to Manganese Chloride Induces Sustained Aberration of Neurogenesis in the Hippocampal Dentate Gyrus of Mice

The effect of exogenously administered manganese (Mn) on developmental neurogenesis in the hippocampal dentate gyrus was examined in male mice after maternal exposure to MnCl(2) (0, 32, 160, or 800 ppm as Mn in diet) from gestational day 10 to day 21 after delivery on weaning. Immunohistochemistry was performed to monitor neurogenesis and interneuron subpopulations on postnatal days (PNDs) 21 and 77 (adult stage). Reelin-synthesizing γ-aminobutyric acid (GABA)ergic interneurons increased in the hilus with ≥ 160 ppm on weaning to sustain to PND 77 at 800 ppm. Apoptosis in the neuroblast-producing subgranular zone increased with 800 ppm and TUC4-expressing immature granule cells decreased with 800 ppm on weaning, whereas at the adult stage, immature granule cells increased. On PND 21, transcript levels increased with Reln and its receptor gene Lrp8 and decreased with Dpysl3 coding TUC4 in the dentate gyrus, confirming immunohistochemical results. Double immunohistochemistry revealed a sustained increase of reelin-expressing and NeuN-lacking or weakly positive immature interneurons and NeuN-expressing mature neurons in the hilus through to the adult stage as examined at 800 ppm. Brain Mn concentrations increased at both PNDs 21 and 77 in all MnCl(2)-exposed groups. These results suggest that Mn targets immature granule cells causing apoptosis and neuronal mismigration. Sustained increases in immature reelin-synthesizing GABAergic interneurons may represent continued aberration in neurogenesis and following migration to cause an excessive response for overproduction of immature granule cells through to the adult stage. Sustained high concentration of Mn in the brain may be responsible for these changes.

Glycidol Induces Axonopathy by Adult-Stage Exposure and Aberration of Hippocampal Neurogenesis Affecting Late-Stage Differentiation by Developmental Exposure in Rats

To investigate the neurotoxicity profile of glycidol and its effect on developmental hippocampal neurogenesis, pregnant Sprague Dawley rats were given drinking water containing 0, 100, 300, or 1000 ppm glycidol from gestational day 6 until weaning on day 21 after delivery. At 1000 ppm, dams showed progressively worsening gait abnormalities, and histopathological examination showed generation of neurofilament-L(+) spheroids in the cerebellar granule layer and dorsal funiculus of the medulla oblongata, central chromatolysis in the trigeminal nerve ganglion cells, and axonal degeneration in the sciatic nerves. Decreased dihydropyrimidinase-like 3(+) immature granule cells in the subgranular zone (SGZ) and increased immature reelin(+) or calbindin-2(+) γ-aminobutyric acid-ergic interneurons and neuron-specific nuclear protein (NeuN)(+) mature neurons were found in the dentate hilus of the offspring of the 1000 ppm group on weaning. Hilar changes remained until postnatal day 77, with the increases in reelin(+) and NeuN(+) cells being present at ≥ 300 ppm, although the SGZ change disappeared. Thus, glycidol caused axon injury in the central and peripheral nervous systems of adult rats, suggesting that glycidol targets the newly generating nerve terminals of immature granule cells, resulting in the suppression of late-stage hippocampal neurogenesis. The sustained hilar changes may be a sign of continued aberrations in neurogenesis and migration. The no-observed-adverse-effect level was determined to be 300 ppm (48.8mg/kg body weight/day) for dams and 100 ppm (18.5mg/kg body weight/day) for offspring. The sustained developmental exposure effect on offspring neurogenesis was more sensitive than the adult axonal injury.

No effect of sustained systemic growth retardation on the distribution of Reelin-expressing interneurons in the neuron-producing hippocampal dentate gyrus in rats

Reelin signaling plays a role in neuronal migration and positioning during brain development. To clarify the effect of systemic growth retardation on the distribution of Reelin-expressing interneurons in the hilus of the hippocampal dentate gyrus, pregnant rats were fed a synthetic diet with either a normal (20% casein) or low (10% casein) protein concentration from gestational day 10 to postnatal day (PND) 21 at weaning. Male offspring were immunohistochemically examined at PND 21 and on PND 77. Protein-restricted offspring displayed systemic growth retardation through PND 77 and had decreased absolute brain weights and an increased number of external granular cells in the cerebellar cortex, suggestive of retarded brain growth at weaning. However, maternal protein restriction did not change the cellular distribution of immunoreactivity for Reelin, Calbindin-D-28K, or glutamic acid decarboxylase 67 or of NeuN-positive postmitotic neurons in the dentate hilus either at PND 21 or PND 77, which suggests that the population of γ-aminobutyric acid-ergic interneurons involving synthesis of Reelin was not affected. Furthermore, as well as the distribution of hilar neurons expressing neurogenesis-related FoxG1, cell proliferation and apoptosis in the subgranular zone were unaffected through PND 77. These results suggest that systemic growth retardation caused by maternal protein restriction does not affect neuronal migration and postnatal neurogenesis of the dentate gyrus resulting in unaltered distribution of Reelin-synthesizing interneurons.

Induction of GST-P-positive proliferative lesions facilitating lipid peroxidation with possible involvement of transferrin receptor up-regulation and ceruloplasmin down-regulation from the early stage of liver tumor promotion in rats

To elucidate the role of metal-related molecules in hepatocarcinogenesis, we examined immunolocalization of transferrin receptor (Tfrc), ceruloplasmin (Cp) and metallothionein (MT)-1/2 in relation to liver cell foci positive for glutathione-S-transferase placental form (GST-P) in the early stage of tumor promotion by fenbendazole (FB), phenobarbital, piperonyl butoxide or thioacetamide in a rat two-stage hepatocarcinogenesis model. To estimate the involvement of oxidative stress responses to the promotion, immunolocalization of 4-hydroxy-2-nonenal, malondialdehyde and acrolein was similarly examined. Our findings showed that MT-1/2 immunoreactivity was not associated with the cellular distribution of GST-P and proliferating cell nuclear antigen, suggesting no role of MT-1/2 in hepatocarcinogenesis. We also found enhanced expression of Tfrc after treatment with strong tumor-promoting chemicals. With regard to Cp, the population showing down-regulation was increased in the GST-P-positive foci in relation to tumor promotion. Up-regulation of Tfrc and down-regulation of Cp was maintained in GST-P-positive neoplastic lesions induced after long-term promotion with FB, suggesting the expression changes occurring downstream of the signaling pathway involved in the formation of GST-P-positive lesions. Furthermore, enhanced accumulation of lipid peroxidation end products was observed in the GST-P-positive foci by promotion. Post-initiation treatment with peroxisome proliferator-activated receptor α agonists did not enhance any such distribution changes in GST-P-negative foci. The results thus suggest that facilitation of lipid peroxidation is involved in the induction of GST-P-positive lesions by tumor promotion from an early stage, and up-regulation of Tfrc and down-regulation of Cp may be a signature of enhanced oxidative cellular stress in these lesions.

Reversible aberration of neurogenesis affecting late-stage differentiation in the hippocampal dentate gyrus of rat offspring after maternal exposure to manganese chloride.

To examine the effects of developmental manganese (Mn)-exposure on hippocampal neurogenesis, pregnant rats were treated with MnCl(2)·4H(2)O in the diet at 32, 160 or 800 ppm from gestation day 10 to day 21 after delivery. Serum concentrations of thyroid-related hormones were examined in offspring exposed to MnCl(2)·4H(2)O at 800 or 1600 ppm. Immunohistochemical analysis revealed increased doublecortin-positive cells in the subgranular zone of the dentate gyrus on postnatal day (PND) 21 following exposure to MnCl(2)·4H(2)O at 800 ppm, indicating an increase of type-3 progenitor or immature granule cells. Reelin-positive cells, suggestive of γ-aminobutyric acid-ergic interneurons in the dentate hilus, also increased at 800 ppm on PND 21. Brain Mn concentrations increased in offspring on PND 21 at 160 and 800 ppm, whereas brain concentrations in the dams were unchanged. Serum concentrations of triiodothyronine and thyroxine decreased at 800 and 1600 ppm, whereas thyroid-stimulating hormone increased only after exposure at 800 ppm. All changes disappeared on PND 77. Thus, maternal exposure to MnCl(2)·4H(2)O at 800 ppm mildly and reversibly affects neurogenesis targeting late-stage differentiation in the hippocampal dentate gyrus of rat offspring. Direct effects of accumulated Mn in the developing brain might be implicated in the mechanism of the development of aberrations in neurogenesis; however, indirect effects through thyroid hormone fluctuations might be rather minor.

Disruptive cell cycle regulation involving epigenetic downregulation of Cdkn2a (p16Ink4a) in early-stage liver tumor-promotion facilitating liver cell regeneration in rats

Cell cycle aberration was immunohistochemically examined in relation to preneoplastic liver cell foci expressing glutathione S-transferase placental form (GST-P) at early stages of tumor-promotion in rats with thioacetamide (TAA), a hepatocarcinogen facilitating liver cell regeneration. Immunoexpression of p16(Ink4a) following exposure to other hepatocarcinogens/promoters and its DNA methylation status were also analyzed during early and late tumor-promotion stages. GST-P(+) liver cell foci increased cell proliferation and decreased apoptosis when compared with surrounding liver cells. In concordance with GST-P(+) foci, checkpoint proteins at G(1)/S (p21(Cip1), p27(Kip1) and p16(Ink4a)) and G(2)/M (phospho-checkpoint kinase 1, Cdc25c and phospho-Wee1) were either up- or downregulated. Cellular distribution within GST-P(+) foci was either increased or decreased with proteins related to G(2)-M phase or DNA damage (topoisomerase IIα, phospho-histone H2AX, phospho-histone H3 and Cdc2). In particular, p16(Ink4a) typically downregulated in GST-P(+) foci and regenerative nodules at early tumor-promotion stage with hepatocarcinogens facilitating liver cell regeneration and in neoplastic lesions at late tumor-promotion stage with hepatocarcinogens/promoters irrespective of regenerating potential. Hypermethylation at exon 2 of Cdkn2a was detected at both early- and late-stages. Thus, diverse disruptive expression of G(1)/S and G(2)/M proteins, which allows for clonal selection of GST-P(+) foci, results in the acquisition of multiple aberrant phenotypes to disrupt checkpoint function. Moreover, increased DNA-damage responses within GST-P(+) foci may be the signature of genetic alterations. Intraexonic hypermethylation may be responsible for p16(Ink4a)-downregulation, which facilitates cell cycle progression in early preneoplastic lesions produced by repeated cell regeneration and late-stage neoplastic lesions irrespective of the carcinogenic mechanism.

Spermatocytic Seminoma With Neuroectodermal Differentiation and Sertoli Cell Tumor in a Dog

Two distinct nodules developed in a cryptorchid testis of an 8-year-old male West Highland White Terrier. One nodule was a Sertoli cell tumor. The other was a spermatocytic seminoma with focal primitive neuroectodermal differentiation: formation of Homer–Wright rosettes and perivascular pseudorosettes, with immunoreactivity for S-100 protein, neuron-specific enolase, synaptophysin, neurofilament–68 kDa, microtubule-associated protein 2, and vimentin. The dog was alive and healthy 2 years after castration.

Reversible effect of maternal exposure to chlorpyrifos on the intermediate granule cell progenitors in the hippocampal dentate gyrus of rat offspring.

To examine the effects of developmental exposure to chlorpyrifos (CPF) on neurogenesis in the hippocampal dentate gyrus, pregnant rats were treated with 2.8, 14 or 70 ppm CPF in the diet from gestational day 10 to day 21 after delivery. Dams had decreased cholinesterase (ChE) activities in red blood cells (RBC) at intakes of ≥2.8 ppm and in brain at 70 ppm. Offspring on postnatal day (PND) 21 had decreased ChE activities in the RBC and brain at 70 ppm. There were no behavioral abnormalities in the offspring. Immunohistochemical analysis showed decreases in the numbers of cells positive for proliferating cell nuclear antigen and T box brain 2 in the subgranular zone (SGZ) of the dentate gyrus on PND 21 at 70 ppm, while other progenitor cell populations and the apoptotic cell number were unaffected in this zone. However, on PND 77 all changes had disappeared. The distribution of the progenitor cell population expressing nicotinic acetylcholine receptor α7 and lacking expression of postmitotic neuron-specific nuclear protein was unchanged by CPF-exposure, suggesting no effect of cholinergic stimulation on neurogenesis. These results suggest that developmental exposure to CPF directly but transiently affect the proliferation of type-2 progenitor cell populations in the hippocampal neurogenesis. The lowest-observed-adverse-effect level (LOAEL) of CPF was determined to be 2.8 ppm (0.36 mg/kg body weight/day) for dams by the inhibition of ChE activity in the RBC at this dose. As for offspring, no-observed-adverse-effect level (NOAEL) was determined to be 14 ppm (1.86 mg/kg body weight/day) by the decrease of type-2 progenitor cell proliferation in the SGZ and the inhibition of ChE activity in the RBC and brain at 70 ppm. The NOAEL of dams based on the offsprings effects was approximately 2800 times higher than the estimated consumption of CPF through food in the general population and in pregnant women as examined in Japan.

Reversible effect of developmental exposure to chlorpyrifos on late-stage neurogenesis in the hippocampal dentate gyrus in mouse offspring

The effect of developmental exposure to chlorpyrifos (CPF) on hippocampal neurogenesis was examined in male mice after maternal dietary exposure to CPF at 0, 4, 20, or 100ppm from gestation day 10 to postnatal day (PND) 21. Cholinesterase activity was dose-dependently decreased in red blood cells at ≥4ppm and in the brain at 100ppm both in dams and offspring on PND 21. Immunohistochemically, doublecortin(+) cells were decreased at ≥20ppm in the subgranular zone (SGZ) of the dentate gyrus, and NeuN(+)-expressing mature neurons were decreased at 100ppm in the hilus on PND 21. There were no differences in the numbers of progenitor populations expressing Tbr2 or M1 muscarinic acetylcholine receptors. Transcript levels of Dcx also decreased at ≥20ppm, and those of Pcna, Casp3, Bax, Bcl2, Pax6 and Tbr2 were unchanged in the dentate gyrus by real-time RT-PCR. At PND 77, hippocampal neurogenesis was unchanged. These results suggest that developmental CPF exposure directly but transiently suppresses maturation of late-stage granule cell lineages in the SGZ and affects interneuron populations in the hilus.

Exposure to MnCl2 · 4H2O during development induces activation of microglial and perivascular macrophage populations in the hippocampal dentate gyrus of rats

Developmental exposure to Mn caused Mn accumulation in the brain tissue and transient disruption of granule cell neurogenesis, targeting the late stage differentiation of progenitor cells in the subgranular zone of the hippocampal dentate gyrus of rats. Because neurogenesis is influenced by proinflammatory responses, this study was performed to determine whether Mn exposure causes microglial activation in the dentate hilus, a region anatomically close to the subgranular zone of the dentate gyrus. Pregnant rats were treated with dietary MnCl2 · 4H2O at 32, 160 or 800 ppm from gestational day 10 to day 21 after delivery. An immunohistochemical analysis revealed increases in Iba1+ microglia in the hilus on postnatal day 21 following exposure to MnCl2 · 4H2O in a dose‐unrelated manner at 32 and at 800 ppm and an increase in CD163+ macrophage at 800 ppm in the hilus. Real‐time reverse transcription–polymerase chain reaction analysis revealed increases in the mRNA levels of Il1α, Il6, Nos2 and Tnf after 800 ppm MnCl2 · 4H2O. These results suggest that activation of microglia and perivascular macrophages occurs in the hilus after developmental exposure to MnCl2 · 4H2O at 800 ppm, and probably involves the disruption of neurogenesis through the accumulation of Mn in the brain tissue. Copyright